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Concept: Pasteurella multocida

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In 2015, more than 200,000 saiga antelopes died in 3 weeks in central Kazakhstan. The proximate cause of death is confirmed as hemorrhagic septicemia caused by the bacterium Pasteurella multocida type B, based on multiple strands of evidence. Statistical modeling suggests that there was unusually high relative humidity and temperature in the days leading up to the mortality event; temperature and humidity anomalies were also observed in two previous similar events in the same region. The modeled influence of environmental covariates is consistent with known drivers of hemorrhagic septicemia. Given the saiga population’s vulnerability to mass mortality and the likely exacerbation of climate-related and environmental stressors in the future, management of risks to population viability such as poaching and viral livestock disease is urgently needed, as well as robust ongoing veterinary surveillance. A multidisciplinary approach is needed to research mass mortality events under rapid environmental change.

Concepts: Physical quantities, Psychrometrics, Future, Pasteurella multocida, Demography, Death, Humidity, Relative humidity

28

Pasteurella multocida is a causative agent of many major diseases of which haemorrhagic septiciemia (HS) in cattle & a buffalo is responsible for significant losses to livestock sector in India and south Asia. The disease outcome is affected by various host- and pathogen-specific determinants. Several bacterial species-specific putative virulence factors including the capsular and virulence associated genes have been proposed to play a key role in this interaction. A total of 23 isolates of P. multocida were obtained from 335 cases of various clinically healthy and diseased cattle. These isolates were examined for capsule synthesis genes (capA, B, D, E and F) and eleven virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) by PCR. A total of 19 P. multocida isolates belonging to capsular type B and 4 of capsular type A were isolated. All isolates of capsular type B harboured the virulence associated genes: tbpA, pfhA, hgbA, sodC and nanH, coding for transferrin binding protein, filamentous hemagglutinin, haemoglobin binding protein, superoxide dismutase and neuraminidases, respectively; while isolates belonging to capsular type A also carried tbpA, pfhA, hgbA and nanH genes. Only 50 % of capsular type A isolates contained sodC gene while 100 % of capsular type B isolates had sodC gene. The gene nanB and toxA were absent in all the 23 isolates. In capsular type A isolates, either sodA or sodC gene was present & these genes did not occur concurrently. The presence of virulence associated gene ptfA revealed a positive association with the disease outcome in cattle and could therefore be an important epidemiological marker gene for characterizing P. multocida isolates.

Concepts: Biology, Epidemiology, Pasteurella multocida, Disease, Gene, Protein, DNA, Infectious disease

27

The antimicrobial properties of florfenicol were investigated for the bovine respiratory tract pathogens, Mannheimia haemolytica and Pasteurella multocida. Three in vitro indices of efficacy and potency were determined; minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and in vitro time-kill curves for six pathogenic strains of each organism. Each was monitored in two matrices, Mueller Hinton broth (MHB) and calf serum. MBC:MIC ratios were low, 1.8 : 1 for M haemolytica in both MHB and serum and 2.4 : 1 and 2.1 : 1 for P multocida in MHB and serum, respectively. The killing action of florfenicol had the characteristics of concentration dependency against M haemolytica and codependency (on time and concentration) against P multocida. Modelling of the time-kill data after 24 hours exposure was undertaken to quantify three levels of activity for the ratio, area under concentration-time curve over 24 hours (AUC24h)/MIC; bacteriostatic action (no change in bacterial count), 3log10 reduction and 4log10 reduction in bacterial count. Mean AUC24h/MIC values for P multocida in MHB (and serum) were 22.0 (23.3) hour, 34.5 (39.9) hour and 45.8 (50.4) hour, respectively. Similar numerical values were obtained for M haemolytica. For both bacterial species, interstrain variability was low; coefficients of variation ( per cent) in serum for 3log10 and 4log10 reductions in count were, respectively, 14.3 and 24.1 for P multocida and 7.8 and 11.4 for M haemolytica. These data form a rational basis for dosage selection for treatment of calf pneumonia caused by M haemolytica or P multocida.

Concepts: Antibiotic, Immune system, Microbiology, Pneumonia, Pasteurella multocida, Ratio, Pathogen, Bacteria

27

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

Concepts: Restriction enzymes, Pasteurella multocida, Enzyme, HindIII, Endonuclease, DNA, Restriction enzyme, Molecular biology

24

The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300 ml phosphate buffered saline (PBS) alone (group 1, n = 3, negative control) or containing 7.1 × 10(8) colony forming units (cfu) of UK isolate (group 2, n = 8) or 5.8 × 10(8) cfu of USA isolate (group 3, n = 8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7-8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4-5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40-41°C within 8-12 h of challenge. Significantly (P = 0.01) greater percentages of lung surface area were consolidated in group 2 (mean ± SD, 21 ± 10.1) compared with group 3 (7 ± 8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P = 0.006) and lymph nodes (P = 0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.

Concepts: Pneumonia, Pulmonology, Cancer, Pasteurellosis, Pasteurella, Touring car racing, Lung, Pasteurella multocida

3

Pasteurella is a Gram-negative coccobacillus that causes a wide spectrum of diseases in humans and is commonly transmitted from cat and dog bites. An increasing number of cats and dogs are kept as pets in American households which increases the risk of pet-related infections.

Concepts: Neutering, Sepsis, Theobromine poisoning, Predation, Cosmopolitan species, Pasteurella multocida, Dog, Septic shock

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Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

Concepts: Pathogen, Assay, Pasteurella multocida, ELISA, Type I and type II errors, Sensitivity and specificity, Microbiology, Bacteria

1

In this retrospective study, 218 pig lung tissue samples were analyzed to examine a possible association between Pneumocystis spp. using in situ hybridization, Bordetella bronchiseptica (B.b.) using immunohistochemistry (IHC), Mycoplasma hyopneumoniae (M.h.) by quantitative PCR, and Pasteurella multocida (P.m.; IHC). Compared to the bacterial agents (B.b., 5%; M.h., 30%; P.m., 23%), Pneumocystis occurred with a higher prevalence (51%). Co-infections with two or three pathogens were present in 28% of the examined cases. Those of Pneumocystis and M.h. were most commonly seen, followed by Pneumocystis and P.m. and M.h. and P.m. Histologically, interstitial pneumonia was found in both the Pneumocystis positive lungs and lungs with a mild M.h. infection. The B.b. and P.m. positive lungs were mainly associated with suppurative bronchopneumonia and severe M.h. cases with fibrinous or fibrino-haemorrhagic pneumonia. In suckling piglets, the number of samples positive for Pneumocystis predominated, whereas samples from fattening pigs were mainly positive for bacteria or Pneumocystis and bacteria.

Concepts: Bordetella, Domestic pig, Pasteurella multocida, Mycoplasma, Pig, Lung, Bacteria, Pneumonia

1

Bovine respiratory disease continues to be the most important ailment of feed yard cattle. While the disease is multifactorial in nature, therapy continues to target the primary bacterial pathogens, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. A survey of records from a single diagnostic laboratory was conducted to evaluate the percentage of M. haemolytica isolates that were resistant to multiple antimicrobials and if coresistance patterns could be detected. All susceptibility test results for M. haemolytica recovered from lung tissues of cattle were eligible for inclusion in the survey. There were no isolates over the course of the analysis that were resistant to all 6 antimicrobials, primarily due to a lack of resistance to ceftiofur. In 2009, just over 5% of isolates were resistant to 5 or more antimicrobials (pan-resistant). In 2011, more than 35% of the M. haemolytica isolates were characterized as pan-resistant. Significant antimicrobial coresistance patterns were only seen with oxytetracycline and tilmicosin; bacterial isolates that were resistant to either oxytetracycline or tilmicosin were more likely to be resistant to at least one other antimicrobial. The mechanisms by which M. haemolytica is developing multidrug resistance warrant investigation if antimicrobial utility in the therapy of bovine respiratory disease is to be preserved.

Concepts: Medicine, Infection, Cancer, Pulmonology, Antibiotic resistance, Pathogen, Pasteurella multocida, Microbiology

0

Hyaluronic acid (HA) is the natural glycosaminoglycan product of HA synthases with diverse cosmetic and medical applications. While process and/or metabolic engineering are utilized for industrial HA production, the potential of protein engineering has barely been realized. Herein, Knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain length specificity and a two-fold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified, eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (up to 4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesized. Computational studies showed that the substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increased the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N-terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Concepts: Amine, N-terminus, Chemical synthesis, Acid, Pasteurella multocida, Metabolism, Amino acid, Protein